Highly Concentrated Bacterial Liquid Soybean Inoculant

ABSTRACT

This invention includes the composition of a highly concentrated liquid inoculant (between 10 10  and 10 11  cfu/mL) for soybean crops. The composition is packed and has a shelf life of up to 2 years stored at temperatures of up to 25° C. The inoculant may be applied in low doses onto seeds or in the furrow.

FIELD OF THE INVENTION

The invention relates to the composition of a liquid inoculant forsoybean, which permits a high concentration of bacteria in a containerstored at room temperature for up to 2 years, and its application at lowdoses.

BACKGROUND OF THE INVENTION

Soybean inoculants use bacteria, usually applied to the seeds or to thesowing furrow, to create a symbiosis with the seedling, and thusleverage the use of nitrogen from the air as a nitrogenous nutrientdirectly provided onto the crop.

The use of inoculants to help the plant transform air nitrogen intousable forms has a strong economic impact on reducing the soybean cropproduction cost, as the use of more expensive synthetic nitrogenfertilizers is avoided. Moreover, the use of biologically-fixed nitrogendoes not have a negative environmental impact, unlike fertilizers with ahighly energy-demanding manufacturing process and their application tothe crop, which can negatively affect the environment.

The production of commercial soybean inoculants has led to productsbased on bacteria Bradyrhizobium japonicum, B. dizoefficiens or B.elkanii contained in different carriers or inerts. These can be of adifferent nature such as peat or clay based powders, or aqueous or oilyliquids. Each provides advantages and disadvantages from the point ofview of bacterial stability and ease of use of the product.

The greatest demand for inoculants based on the type of formulationcarrier is for liquids, preferably aqueous ones. These types ofinoculants provide convenience of application both to the seed and tothe sowing furrow and are adapted to many situations and treatmentvariants.

Commercial liquid inoculants generally have a shelf life of 6 to 18months and their storage temperature ranges between 5 and 25° C. Storageand transport must occur away from solar radiation.

When treating seeds, the use of an inoculant together with phytosanitaryproducts and other chemical or biological products is very common, sincethe user needs to ensure the correct establishment of the crop. Productssuch as fungicides, insecticides, nematicides, micronutrients, polymers,among others, minimize the risk of phytopathogens and insects attack,and provide excellent seed coverage, thus achieving homogeneity ofapplication, proper movement and a good aspect.

However, from the point of view of the seed integrity, there is a limitto the amount of products to be applied onto it, beyond which problemssuch as deterioration, germination and adhesiveness between seeds mayoccur. The maximum volume of liquid that can be tolerated by soybeanwithout generating inconveniences is approximately 5 mL/kg for a singlesimultaneous application and 10 mL/kg for the sum of sequentialapplications. The seed grower should apply all relevant products withoutexceeding this volume. It is here when the use the lowest possible dosesof products while maintaining their effectiveness to minimize the risksof seed deterioration becomes vital.

The available soybean inoculants on the market are applied at relativelyhigh doses (approximately 2-3 mL/kg) and several of them involve the useof a bacterial protector (approximately 0.5-2 mL/kg) which contributesto increasing the total dose of products intended to leverage nitrogenfrom the air. These amounts of inoculant and protection plus the otherproducts in a typical seed treatment may exceed the maximum recommendedliquid limit and, therefore, increase the risks of failure (biologicaland/or physical). Furthermore, highly concentrated products may beavailable in paste form. However, they require refrigerated storage andan activation process before application, which affects their cost andpracticality of use. This is why they are generally used as an input forother types of inoculants and not as an end product.

Besides contributing to the efficiency of seed treatment, low doses ofinoculant result in a reduction of transportation cost, storage space,energy consumed during application, and general handling. In addition,they result in a positive impact on the environment due to the reductionof the amount of packaging material used per seed weight unit.

Consequently, there is a need for a soybean inoculant composition with ahigh concentration of bacterial count per volume unit (between 10¹⁰ and10¹¹ cfu/mL), which can be stored and transported under normal roomtemperature conditions for inoculants in general, which maintains itseffectiveness over a long shelf life in its packaging, which is liquidin order to maximize its practicality of use, and which is applied atrelatively low doses so that it can be applied together with otherproducts.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to a composition of a liquid inoculant forsoybean, with a high concentration of bacteria per volume unit (between10¹⁰ and 10¹¹ cfu/mL), with a shelf life of up to 2 years at roomtemperature, and with a minimum concentration at expiry of 2×10¹⁰cfu/mL. The composition includes providing the elements of itsformulation, preparation and packaging.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows B. japonicum survival in a highly concentrated liquidinoculant of different compositions and manufacturing processes based onstorage time at room temperature (20-25° C.).

FIG. 2 shows B. japonicum survival in a highly concentrated liquidinoculant of two compositions according to the type of stabilizer, basedon storage time at room temperature (20-25° C.).

FIG. 3 shows B. japonicum survival in a highly concentrated liquidinoculant, based on storage time at two temperatures (16 and 20-25° C.).

FIG. 4 shows B. japonicum survival in a highly concentrated liquidinoculant, according to the degree of concentration of the bacterialbroth culture and the proportion of the stabilizer used in the finalformulation, based on storage time at room temperature (20-25° C.).

FIG. 5 shows the number of Bradyrhizobium colony forming units (cfu) perseed, when the highly concentrated liquid inoculant is applied after twoyears of storage.

FIG. 6 shows the number of nodules per plant in primary root, when thehighly concentrated liquid inoculant is applied after two years ofstorage.

FIG. 7 shows the number of nodules per plant in total root, when thehighly concentrated liquid inoculant is applied after two years ofstorage.

FIG. 8 shows the dry mass of nodules per plant in primary root, when thehighly concentrated liquid inoculant is applied after two years ofstorage.

FIG. 9 shows the dry mass of nodules per plant in total root, when thehighly concentrated liquid inoculant is applied after two years ofstorage.

DETAILED DESCRIPTION OF THE INVENTION AND DRAWINGS

Reference is made to the composition of a liquid inoculant highlyconcentrated in bacteria of the Bradyrhizobium genus that nodulatesoybean plants, such as: B. japonicum; B. diazoefficiens; B. elkanii.The composition adapts to the usual commercial handling of inoculantsunder the usual storage and transport conditions, and it can reach ashelf life of up to two years and be applied at low doses.

Phase 1—Cellular Multiplication

Firstly, Bradyrhizobium bacteria, such as B. japonicum; B.diazoefficiens; B. elkanii, are multiplied in an appropriate culturemedium such as the widely reported YMB (Yeast-Mannitol-Broth) or itsderivations, such as the medium shown in Table 1, which is preferred forthe composition of the highly concentrated inoculant to which thisinvention relates.

TABLE 1 Modified YMB medium - g/L Glycerol 25 Yeast Extract 4 KNO₃ 1.6MgSO₄•7H₂O 0.2 K₂HPO₄ 0.5 KH₂PO₄ 0.5 (NH₄)₂HPO₄ 0.4 NaCl 0.1 MnSO₄•H₂O0.01 FeCl₃ 0.01 Water 1000 pH: 6.8-7.0

Bacteria are added to the culture medium, leaving an initialconcentration of 1×10⁸ to 1×10⁹ cfu/mL, preferably 3×10⁸ to 7×10⁸cfu/mL. Under appropriate temperature and agitation conditions, cellularmultiplication suitable for this first stage of processing of the highlyconcentrated inoculant composition is obtained.

The appropriate incubation temperature ranges between 23 and 30° C.,preferably between 27 and 30° C., and the degree of aeration is variableand depends on the element where the cellular multiplication takesplace, ranging from Erlenmeyer flasks in an agitation incubator tobio-reactors or automated fermenters. Incubation time can range from 4to 7 days depending on the bacterial strain considered within thespecies mentioned in this invention.

At the end of incubation, a level of bacterial concentration between1.5×10¹⁰ to 3×10¹⁰ cfu/mL must be reached. This first stage of bacterialculture development is followed by Phase 2. which includes the processof centrifugation or cell separation from its surrounding medium, withthe aim of achieving a highly concentrated bacterial broth ready tocontinue with the last phase (Phase 3) involving the final formulationprocess and packaging.

Phase 2—Cellular Concentration

The centrifugation or cell separation process concentrates the amount ofbacteria in the bacterial broth from Phase 1. A laboratory centrifuge oran industrial separator can be used for this purpose. For the first casethe proper centrifugation speed is between 5000 and 14000 rpm, andspeeds between 8000 and 12000 rpm are preferred. Centrifugation time andtemperature can vary between 5 and 20 minutes and between 18 and 26° C.,respectively, preferably 8 to 15 minutes and 20 to 25° C.

Bacterial broth concentration means that from a normal bacterial broth abacterial broth concentrated 8 to 20 times is obtained. To use theconcentrated broth within the mentioned range, the sediment and part ofthe supernatant liquid must be used. The sum of the sediment volume andthe supernatant volume represents the desired concentration within thementioned range. The sediment is then vigorously mixed with theremaining supernatant and a homogeneous re-suspension of the highlyconcentrated bacterial broth is obtained.

This material is then ready for final formulation and packaging process(Phase 3), which will result in the highly concentrated inoculant.

Phase 3—Final Formulation

Once the 8× to 20× concentrated bacterial broth is obtained, the finalformulation stabilizes the product to achieve a liquid composition witha shelf life of up to 2 years under storage conditions at roomtemperature of 20-25° C. The parameter defining the shelf life is thebacterial concentration ranging between 10¹⁰ and 10¹¹ cfu/mL, morespecifically between 2×10¹⁰ and 2×10¹¹ cfu/mL, capable of producingoptimal nodulation in the soybean crop.

The formulation consists of adding one or more substances mixed withwater to the highly concentrated bacterial broth. The substances,dissolved in water, produce “stabilizing” options which can be added tothe highly concentrated bacterial broth (Table 2). After the stabilizeris added to the broth, it is exposed to interaction under the sametemperature and agitation conditions as in Phase 1.

The stabilizer used must be compatible with the viability of thebacteria and, at the same time, protect the cells from prematuredeterioration both in the container and on the seed or in the soil.

TABLE 2 Substance Substance concentration in Sucrose Trehalose CMCK₂HPO₄ KH₂PO₄ Water the Stabilizer (% Stabilizer (g) (g) (g) (g) (g)(mL) weight/weight) A1 2000 0 0 5 2.5 1000 66.7 A1810 1275 0 6.3 5 2.51000 56.3 A2 0 1000 0 5 2.5 1000 50.2

The stabilizer is incorporated proportionately ranging from 10 to 75%with respect to the volume of the highly concentrated bacterial broth.Preferably, the proportions used range between 20 and 50%.

Once the highly concentrated and stabilizing bacterial broth mixture isobtained, the formulation is subjected to an interaction time called“Process” (P) under the same temperature and agitation conditions as inPhase 1. Processes can vary between 0 and 5 days, preferably 2 to 4days. The Process may increase the physicochemical assimilation of thestabilizer into the cells and may contribute to improve thestabilization and cell protection qualities of the highly concentratedliquid inoculant.

Once the formulation of the highly concentrated liquid inoculant iscomplete, it is packaged. The composition can be placed in single- ordouble-layer low density polyethylene bladders, each layer 35 to 80 μmthick, preferably 50 to 70 μm, using a volume of 0.5 to 15 L, preferably0.5 to 2 L. This type of packaging allows the gaseous exchange betweenthe composition and the environment, contributing to the metabolicbalance of the bacteria and their stabilization during the product'slife. In addition, it maintains purity and prevents contamination ofparticles inside.

The packaged product can be stored at temperatures between 2 to 25° C.,although the composition design allows temperatures between 20 to 25° C.while maintaining its useful life for up to 2 years. Given the type ofpackaging used, no specific relative air humidity is required. There areno specific requirements for brightness either, although it should bepreferably stored in the dark.

EXAMPLES Example 1

Survival of Bradyrhizobium japonicum on Soybean and Nodulation Responsein Treatments with Agrochemicals Used in Brazil

The following example shows the results of the use of the highlyconcentrated inoculant after storage for 1.5 years from its manufacture.The effect of two preservation temperatures of the treated seed wasevaluated (20-24° C. and 26° C.). In addition, it was applied withdifferent commercial agrochemicals commonly used in Brazil and itsperformance was evaluated. The highly concentrated inoculant was appliedin 2 different doses and using different application methods. Theresults were evaluated: the viability of the bacteria on the soybean wasmeasured, the number of bacteria per seed were counted, and thenodulation response in the plant growth chamber was observed, accordingto the time since the treatment (pre-inoculation).

The highly concentrated liquid inoculant used in this example wasmanufactured from Stabilizer A1 and contained 1.65×10¹¹ cfu/mL, with 1.5year since its manufacture. Prior to application on soybeans, theinoculant (UHC) was mixed with a bacterial protector (Premax LLI). Themixture was let sit for 15 minutes and the proper dose was applied to200 g of DM 4014 soybean which had been previously treated withcommercial agrochemicals. It was also considered a control withoutagrochemicals. Table 3 shows the products that were used and theirapplication rates on the seeds.

TABLE 3 Dosage (quantity/kg Product name Product type Active ingredient(%) soybean seed) UHC Highly Bradyrhizobium japonicum + 3 mL (dose1)concentrated Bradyrhizobium diazoefficiens (15) or 4 mL bacterial(dose2) inoculant Premax LLI Bacterial Sugar (% not applicable) 1 mLprotector Avicta 500 FS Nematicide and Abamectine (50) 1 mL insecticideCruiser 35 FS Insecticide Thiamethoxam (35) 2 mL Maxim FungicideTiabendazole (15) + 1 mL Evolution fludioxonil (2.5) + metalaxyl-M (2)Fluidus 028 Drying powder Micas (% not applicable) 1 g AmuletInsecticide Fipronil (20) 1 mL Cropstar Insecticide Imidacloprid (15) +thiodicarb (45) 6 mL Ritiram Carb Fungicide Thiram (35) + carbendazim(15) 2 mL Plus Standak Top Fungicide and Pyraclostrobin (2.5) + Methyl 2mL insecticide thiophanate (22.5) + Fipronil (25) Dermacor InsecticideChlorantraniliprole (62.5) 1 mL Seed + Dry Nutrition Ca (4.2) + Mg(1.9) + S (4.2) + Co 2.5 g supplement (0.0012) + Cu (0.08) + Fe (0.9) +Mn (0.25) + Mo (0.0008) + Zn (1)

Levels 34, 63 and 78 days after inoculation (dai) were established,which are the times between seed treatment and the biologicaldeterminations (early seed treatment or pre-inoculation). The treatedseed was stored at two temperatures (20-24° C. and 26° C.) and indarkness.

Technics and Measurements of Results

The number of viable Bradyrhizobium per seed was determined using thetechnique of decimal dilutions and surface dissemination (0.1 mL ofinoculum) in Petri dishes containing YMA (Yeast-Mannitol-Agar) medium.In each determination, 50 seeds were shaken for 12 min with 50 mL ofpurified (demineralized) water sterilized by autoclave, in a 125 mLbottle, at 200 rpm. Three repetitions and 2 dishes by dilution wereused. The sown dishes were incubated at 29.5° C. and Bradyrhizobiumcolonies were counted 7 days later.

For the nodulation study, seed was sown in cups with 330 g of sandy soilfree of Bradyrhizobium (from La Pampa, Argentina, with no soybean crophistory). The units were placed in a growth chamber under controlledconditions of temperature (25-28° C.), soil moisture (close to fieldcapacity) and air (60-70% RH), light (8000 lux) and photoperiod (16 hlight, 8 h darkness). Irrigation was carried out with purified(demineralized) water sterilized by filtration (0.1 μm). Two seeds perglass were sown and on the fourth day a thinning of 1 plant per glasswas done. We worked with 9 repetitions and 35 days after sowing theplants were processed to determine the 4 nodulation variables: number ofnodules per plant (by direct count) in primary and total root; and drymass of nodules per plant (by weighing nodules dried at 60° C. for 48hours) in primary and total root.

The response of the treatments can be evaluated according to benchmarksas suitability or agronomic efficiency indicators. One of thesebenchmarks may be one that meets the minimum acceptable values of two ormore of the variables analyzed: Bradyrhizobium cfu/seed, ≥5×10⁴; Numberof nodules per plant in primary root ≥2; Number of nodules per plant intotal root ≥3; dry mass (mg) of nodules per plant in primary root ≥10;and dry mass (mg) of nodules per plant in total root ≥15. In order todetermine suitability or efficiency, the following variables arepreferably taken into account: number of nodules per plant in total rootand dry mass (mg) of nodules per plant in total root.

Results

Table 4 shows that the highly concentrated (UHC) bacterial inoculant wasefficient, as it responded to several cases with ≥3 nodules per plant intotal root and ≥15 mg of dry mass of nodules in total root. Suitabilitywas influenced by the degree of compatibility with agrochemicals, seedstorage temperature, pre-inoculation time and inoculant dose. A suitableresponse was achieved of up to 78 days after inoculation (dai) in thepresence of different combinations of agrochemicals and seed storage at20-24° C. The best response under these conditions was observed in seedtreatments with “Maxim Evolution+Standak Top//UHC+Premax LLI dose1”,“Avicta 500 FS+Cruiser 35 FS+Maxim Evolution//UHC+Premax LLI dose2” and“UHC+Premax LLI dose2/Fluidus 028”, which produced ≥6 nodules per plantin total root and ≥32.3 mg of dry mass of nodules in total root.

TABLE 4 Dry Dry mass mass No of No of (mg) (mg) Seed nodules/ nodules/nodules/ nodules/ storage plant in plant in plant in plant intemperature Seed cfu/ Primary Total Primary Total dai (° C.) treatmentseed Root Root Root Root 34 20-24 UHC + Premax LLI 5.72E+05 4.6 6.9 48.360.0 dose1 34 20-24 Avicta 500 FS + 3.08E+05 4.4 18.5 35.9 67.1 Cruiser35 FS + Maxim Evolution // UHC + Premax LLI dose1 34 20-24 Avicta 500FS + 1.97E+05 1.6 11.3 19.6 56.6 Cruiser 35 FS + Maxim Evolution //UHC + Premax LLI dose1/Fluidus 028 34 20-24 Amulet + Cruiser 35 1.15E+053.6 13.1 33.2 63.3 FS + Maxim Evolution // UHC + Premax LLI dose1 3420-24 Cropstar // UHC + 4.75E+03 0.0 0.9 0.0 5.0 Premax LLI dose1 3420-24 Maxim Evolution + 2.77E+05 0.0 3.3 0.0 17.8 Ritiram Carb Plus //UHC + Premax LLI dose1 34 20-24 Maxim Evolution + 1.95E+05 0.0 3.3 0.013.8 Ritiram Carb Plus // UHC + Premax LLI dose1/Fluidus 028 34 20-24Maxim Evolution + 1.04E+06 1.4 15.6 9.8 53.4 Standak Top // UHC + PremaxLLI dose1 34 20-24 Dermacor // UHC + 7.77E+05 2.1 14.1 19.9 49.0 PremaxLLI dose1 34 20-24 UHC + Premax LLI 4.57E+05 1.0 14.7 10.4 49.4dose1/Fluidus 028 34 20-24 Standak Top // UHC + 3.90E+05 2.9 9.0 27.248.9 Premax LLI dose1 34 20-24 UHC + Premax LLI 9.17E+04 0.8 4.2 6.828.3 dose1/Seed + Dry 34 20-24 UHC + Premax LLI 1.79E+06 2.3 15.9 30.655.0 dose2 34 20-24 Avicta 500 FS + 1.05E+06 2.7 12.1 29.2 63.2 Cruiser35 FS + Maxim Evolution // UHC + Premax LLI dose2 34 20-24 Avicta 500FS + 6.68E+05 3.8 11.2 26.9 55.3 Cruiser 35 FS + Maxim Evolution //UHC + Premax LLI dose2/Fluidus 028 34 20-24 Amulet + Cruiser 35 7.50E+054.2 15.2 37.0 71.1 FS + Maxim Evolution // UHC + Premax LLI dose2 3420-24 Cropstar // UHC + 9.00E+03 0.0 1.0 0.0 10.2 Premax LLI dose2 3420-24 Maxim Evolution + 1.04E+06 0.0 3.2 0.0 22.4 Ritiram Carb Plus //UHC + Premax LLI dose2 34 20-24 Maxim Evolution + 2.42E+05 0.0 3.8 0.023.8 Ritiram Carb Plus // UHC + Premax LLI dose2/Fluidus 028 34 20-24Maxim Evolution + 1.14E+06 1.1 17.7 11.7 60.9 Standak Top // UHC +Premax LLI dose2 34 20-24 Dermacor // UHC + 1.57E+06 4.2 7.8 48.4 57.9Premax LLI dose2 34 20-24 UHC + Premax LLI 9.87E+05 2.2 10.2 25.2 54.8dose2/Fluidus 028 34 20-24 Standak Top // UHC + 6.18E+05 6.4 13.8 48.065.2 Premax LLI dose2 34 20-24 UHC + Premax LLI 1.20E+05 0.2 5.2 5.226.7 dose2/Seed + Dry 34 26 UHC + Premax LLI 3.17E+05 3.2 13.2 39.4 54.2dose1 34 26 Avicta 500 FS + 2.67E+05 1.8 13.9 17.4 57.6 Cruiser 35 FS +Maxim Evolution // UHC + Premax LLI dose1 34 26 Avicta 500 FS + 7.60E+040.8 6.2 7.0 38.0 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLIdose1/Fluidus 028 34 26 Amulet + Cruiser 35 1.15E+05 0.0 6.9 0.0 46.9FS + Maxim Evolution // UHC + Premax LLI dose1 34 26 Cropstar // UHC +8.33E+03 0.0 1.0 0.0 8.1 Premax LLI dose1 34 26 Maxim Evolution +8.17E+04 0.0 3.2 0.0 10.8 Ritiram Carb Plus // UHC + Premax LLI dose1 3426 Maxim Evolution + 1.38E+05 0.0 0.8 0.0 4.7 Ritiram Carb Plus // UHC +Premax LLI dose1/Fluidus 028 34 26 Maxim Evolution + 1.50E+05 0.1 10.10.7 49.6 Standak Top // UHC + Premax LLI dose1 34 26 Dermacor // UHC +1.28E+05 0.4 7.6 7.3 36.7 Premax LLI dose1 34 26 UHC + Premax LLI 124E+05 0.6 10.9 6.8 50.8 dose1/Fluidus 028 34 26 Standak Top // UHC +8.27E+04 0.0 7.9 0.0 49.2 Premax LLI dose1 34 26 UHC + Premax LLI2.08E+04 0.0 0.9 0.0 10.2 dose1/Seed + Dry 34 26 UHC + Premax LLI7.28E+05 2.7 6.9 38.3 52.3 dose2 34 26 Avicta 500 FS + 1.43E+05 0.1 8.81.1 49.8 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose2 34 26Avicta 500 FS + 1.07E+05 0.1 5.8 1.8 41.2 Cruiser 35 FS + MaximEvolution // UHC + Premax LLI dose2/Fluidus 028 34 26 Amulet + Cruiser35 1.02E+05 0.3 10.6 6.2 46.7 FS + Maxim Evolution // UHC + Premax LLIdose2 34 26 Cropstar // UHC + 6.00E+03 0.0 0.1 0.0 1.1 Premax LLI dose234 26 Maxim Evolution + 5.38E+04 0.0 0.6 0.0 7.1 Ritiram Carb Plus //UHC + Premax LLI dose2 34 26 Maxim Evolution + 5.40E+04 0.0 0.4 0.0 4.9Ritiram Carb Plus // UHC + Premax LLI dose2/Fluidus 028 34 26 MaximEvolution + 1.91E+05 0.3 13.9 3.8 59.2 Standak Top // UHC + Premax LLIdose2 34 26 Dermacor // UHC + 1.88E+05 1.9 9.0 18.3 45.6 Premax LLIdose2 34 26 UHC + Premax LLI 3.48E+05 1.3 6.7 20.4 48.2 dose2/Fluidus028 34 26 Standak Top // UHC + 1.17E+05 1.1 9.1 11.3 56.0 Premax LLIdose2 34 26 UHC + Premax LLI 4.33E+04 0.2 2.9 3.4 16.4 dose2/Seed + Dry63 20-24 UHC + Premax LLI 7.58E+04 1.4 10.3 18.6 35.1 dose1 63 20-24Avicta 500 FS + 3.72E+04 0.0 11.0 0.0 51.2 Cruiser 35 FS + MaximEvolution // UHC + Premax LLI dose1 63 20-24 Avicta 500 FS + 2.40E+040.3 5.4 6.5 40.0 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLIdose1/Fluidus 028 63 20-24 Amulet + Cruiser 35 8.50E+03 0.0 7.3 0.0 40.3FS + Maxim Evolution // UHC + Premax LLI dose1 63 20-24 Cropstar //UHC + 3.65E+04 0.0 0.2 0.0 1.0 Premax LLI dose1 63 20-24 MaximEvolution + 1.50E+04 0.0 0.4 0.0 2.1 Ritiram Carb Plus // UHC + PremaxLLI dose1 63 20-24 Maxim Evolution + 3.83E+03 0.0 0.4 0.0 1.7 RitiramCarb Plus // UHC + Premax LLI dose1/Fluidus 028 63 20-24 MaximEvolution + 3.17E+04 0.1 5.7 3.0 40.3 Standak Top // UHC + Premax LLIdose1 63 20-24 Dermacor // UHC + 4.25E+04 0.3 4.4 8.6 23.8 Premax LLIdose1 63 20-24 UHC + Premax LLI 6.50E+04 1.0 5.8 15.7 42.3 dose1/Fluidus028 63 20-24 Standak Top // UHC + 1.00E+04 0.0 4.4 0.0 30.6 Premax LLIdose1 63 20-24 UHC + Premax LLI 1.50E+04 0.0 2.1 0.0 14.7 dose1/Seed +Dry 63 20-24 UHC + Premax LLI 1.47E+05 1.8 7.0 23.9 48.8 dose2 63 20-24Avicta 500 FS + 7.95E+04 0.0 7.0 0.0 50.3 Cruiser 35 FS + MaximEvolution // UHC + Premax LLI dose2 63 20-24 Avicta 500 FS + 6.83E+040.0 2.9 0.0 18.2 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLIdose2/Fluidus 028 63 20-24 Amulet + Cruiser 35 3.83E+04 0.3 6.0 5.3 43.7FS + Maxim Evolution // UHC + Premax LLI dose2 63 20-24 Cropstar //UHC + 2.00E+03 0.0 0.3 0.0 2.1 Premax LLI dose2 63 20-24 MaximEvolution + 2.67E+04 0.0 0.0 0.0 0.0 Ritiram Carb Plus // UHC + PremaxLLI dose2 63 20-24 Maxim Evolution + 7.50E+03 0.9 4.9 10.6 39.4 RitiramCarb Plus // UHC + Premax LLI dose2/Fluidus 028 63 20-24 MaximEvolution + 2.00E+04 0.2 3.6 3.4 23.7 Standak Top // UHC + Premax LLIdose2 63 20-24 Dermacor // UHC + 4.50E+04 0.0 4.8 0.0 30.0 Premax LLIdose2 63 20-24 UHC + Premax LLI 1.05E+05 0.8 2.7 11.8 24.2 dose2/Fluidus028 63 20-24 Standak Top // UHC + 4.50E+03 0.2 6.6 3.2 43.2 Premax LLIdose2 63 20-24 UHC + Premax LLI 2.33E+04 0.0 0.7 0.0 1.8 dose2/Seed +Dry 63 26 UHC + Premax LLI 1.65E+04 0.2 4.9 4.1 32.6 dose1 63 26 Avicta500 FS + 3.50E+03 0.0 4.3 0.0 26.1 Cruiser 35 FS + Maxim Evolution //UHC + Premax LLI dose1 63 26 Avicta 500 FS + 1.33E+03 0.0 1.4 0.0 9.2Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose1/Fluidus 028 6326 Amulet + Cruiser 35 2.17E+03 0.0 2.6 0.0 18.1 FS + Maxim Evolution //UHC + Premax LLI dose1 63 26 Cropstar // UHC + 1.00E+03 0.0 0.0 0.0 0.0Premax LLI dose1 63 26 Maxim Evolution + 2.25E+03 0.0 0.0 0.0 0.0Ritiram Carb Plus // UHC + Premax LLI dose1 63 26 Maxim Evolution +1.00E+03 0.0 0.4 0.0 0.3 Ritiram Carb Plus // UHC + Premax LLIdose1/Fluidus 028 63 26 Maxim Evolution + 2.00E+03 0.0 0.9 0.0 4.9Standak Top // UHC + Premax LLI dose1 63 26 Dermacor // UHC + 1.75E+030.0 1.3 0.0 9.4 Premax LLI dose1 63 26 UHC + Premax LLI 1.33E+04 0.0 4.00.0 17.4 dose1/Fluidus 028 63 26 Standak Top // UHC + 1.00E+03 0.0 1.40.0 22.6 Premax LLI dose1 63 26 UHC + Premax LLI 2.25E+03 0.0 0.4 0.02.1 dose1/Seed + Dry 63 26 UHC + Premax LLI 2.17E+04 1.1 6.1 23.6 45.0dose2 63 26 Avicta 500 FS + 4.00E+03 0.1 2.2 1.9 21.7 Cruiser 35 FS +Maxim Evolution // UHC + Premax LLI dose2 63 26 Avicta 500 FS + 2.67E+030.0 1.2 0.0 8.4 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLIdose2/Fluidus 028 63 26 Amulet + Cruiser 35 3.50E+03 0.0 1.7 0.0 10.4FS + Maxim Evolution // UHC + Premax LLI dose2 63 26 Cropstar // UHC +1.00E+03 0.0 0.1 0.0 1.1 Premax LLI dose2 63 26 Maxim Evolution +1.00E+03 0.0 0.3 0.0 2.0 Ritiram Carb Plus // UHC + Premax LLI dose2 6326 Maxim Evolution + 1.75E+03 0.0 0.0 0.0 0.0 Ritiram Carb Plus // UHC +Premax LLI dose2/Fluidus 028 63 26 Maxim Evolution + <1.00E+03  0.0 2.30.0 14.3 Standak Top // UHC + Premax LLI dose2 63 26 Dermacor // UHC +2.50E+03 0.0 0.7 0.0 11.6 Premax LLI dose2 63 26 UHC + Premax LLI4.28E+04 0.0 3.8 0.0 21.6 dose2/Fluidus 028 63 26 Standak Top // UHC +1.80E+04 0.0 2.4 0.0 19.4 Premax LLI dose2 63 26 UHC + Premax LLI1.35E+04 0.0 0.8 0.0 5.1 dose2/Seed + Dry 78 20-24 UHC + Premax LLI1.62E+04 0.0 6.0 0.0 27.1 dose1 78 20-24 Avicta 500 FS + 1.03E+04 0.03.9 0.0 28.1 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose178 20-24 Avicta 500 FS + 4.33E+03 0.0 0.4 0.0 6.3 Cruiser 35 FS + MaximEvolution // UHC + Premax LLI dose1/Fluidus 028 78 20-24 Amulet +Cruiser 35 5.67E+03 0.1 1.1 1.6 13.4 FS + Maxim Evolution // UHC +Premax LLI dose1 78 20-24 Cropstar // UHC + <1.00E+03  0.0 0.4 0.0 2.9Premax LLI dose1 78 20-24 Maxim Evolution + 2.00E+03 0.0 0.5 0.0 3.8Ritiram Carb Plus // UHC + Premax LLI dose1 78 20-24 Maxim Evolution +2.17E+03 0.0 0.8 0.0 3.0 Ritiram Carb Plus // UHC + Premax LLIdose1/Fluidus 028 78 20-24 Maxim Evolution + 6.00E+03 2.4 6.6 19.6 32.3Standak Top // UHC + Premax LLI dose1 78 20-24 Dermacor // UHC +5.67E+03 1.4 3.3 4.4 8.4 Premax LLI dose1 78 20-24 UHC + Premax LLI1.73E+04 2.0 6.3 14.1 28.3 dose1/Fluidus 028 78 20-24 Standak Top //UHC + 1.38E+04 1.4 5.4 3.9 16.4 Premax LLI dose1 78 20-24 UHC + PremaxLLI 2.83E+03 0.0 0.6 0.0 1.1 dose1/Seed + Dry 78 20-24 UHC + Premax LLI5.40E+04 2.4 8.4 23.5 41.8 dose2 78 20-24 Avicta 500 FS + 1.50E+04 1.06.0 7.6 32.8 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose278 20-24 Avicta 500 FS + 7.83E+03 1.5 5.3 9.5 23.9 Cruiser 35 FS + MaximEvolution // UHC + Premax LLI dose2/Fluidus 028 78 20-24 Amulet +Cruiser 35 1.25E+04 1.5 3.5 15.6 24.6 FS + Maxim Evolution // UHC +Premax LLI dose2 78 20-24 Cropstar // UHC + 1.00E+03 0.1 0.8 6.0 9.4Premax LLI dose2 78 20-24 Maxim Evolution + 3.67E+03 0.1 0.3 0.5 0.6Ritiram Carb Plus // UHC + Premax LLI dose2 78 20-24 Maxim Evolution +7.50E+03 0.1 0.8 0.1 1.5 Ritiram Carb Plus // UHC + Premax LLIdose2/Fluidus 028 78 20-24 Maxim Evolution + 5.67E+03 0.8 3.1 4.5 14.1Standak Top // UHC + Premax LLI dose2 78 20-24 Dermacor // UHC +8.17E+03 0.3 2.9 7.1 13.6 Premax LLI dose2 78 20-24 UHC + Premax LLI2.17E+04 2.0 6.1 31.0 39.4 dose2/Fluidus 028 78 20-24 Standak Top //UHC + 7.50E+03 0.4 3.3 5.6 22.6 Premax LLI dose2 78 20-24 UHC + PremaxLLI 3.50E+03 0.1 1.6 0.1 4.8 dose2/Seed + Dry 78 26 UHC + Premax LLI4.93E+03 0.0 1.4 0.0 7.4 dose1 78 26 Avicta 500 FS + 3.17E+02 0.0 1.80.0 12.4 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose1 78 26Avicta 500 FS + 3.00E+02 0.0 0.1 0.0 0.8 Cruiser 35 FS + Maxim Evolution// UHC + Premax LLI dose1/Fluidus 028 78 26 Amulet + Cruiser 35 1.83E+020.0 0.3 0.0 5.5 FS + Maxim Evolution // UHC + Premax LLI dose1 78 26Cropstar // UHC + <1.00E+02  0.0 0.1 0.0 0.8 Premax LLI dose1 78 26Maxim Evolution + 2.00E+02 0.0 0.0 0.0 0.0 Ritiram Carb Plus // UHC +Premax LLI dose1 78 26 Maxim Evolution + 1.05E+03 0.0 0.5 0.0 0.4Ritiram Carb Plus // UHC + Premax LLI dose1/Fluidus 028 78 26 MaximEvolution + 2.25E+02 0.0 0.8 0.0 3.9 Standak Top // UHC + Premax LLIdose1 78 26 Dermacor // UHC + 1.95E+03 0.0 0.1 0.0 0.5 Premax LLI dose178 26 UHC + Premax LLI 5.67E+02 0.3 1.1 1.8 3.8 dose1/Fluidus 028 78 26Standak Top // UHC + 3.83E+02 0.4 0.9 3.4 4.9 Premax LLI dose1 78 26UHC + Premax LLI 2.17E+02 0.0 0.1 0.0 0.1 dose1/Seed + Dry 78 26 UHC +Premax LLI 2.78E+03 0.8 2.0 2.9 10.9 dose2 78 26 Avicta 500 FS +1.50E+03 0.8 2.3 11.5 18.3 Cruiser 35 FS + Maxim Evolution // UHC +Premax LLI dose2 78 26 Avicta 500 FS + 3.50E+02 0.1 1.8 0.6 5.4 Cruiser35 FS + Maxim Evolution // UHC + Premax LLI dose2/Fluidus 028 78 26Amulet + Cruiser 35 3.00E+02 0.0 0.9 0.0 3.5 FS + Maxim Evolution //UHC + Premax LLI dose2 78 26 Cropstar // UHC + 1.00E+02 0.0 0.5 0.0 1.9Premax LLI dose2 78 26 Maxim Evolution + 1.00E+02 0.0 0.3 0.0 0.1Ritiram Carb Plus // UHC + Premax LLI dose2 78 26 Maxim Evolution +2.00E+02 0.0 0.8 0.0 1.8 Ritiram Carb Plus // UHC + Premax LLIdose2/Fluidus 028 78 26 Maxim Evolution + 3.00E+02 0.0 0.4 0.0 1.8Standak Top // UHC + Premax LLI dose2 78 26 Dermacor // UHC + 1.00E+020.0 0.0 0.0 0.0 Premax LLI dose2 78 26 UHC + Premax LLI 1.25E+03 0.0 0.30.0 0.9 dose2/Fluidus 028 78 26 Standak Top // UHC + <1.00E+02  0.4 1.05.4 11.0 Premax LLI dose2 78 26 UHC + Premax LLI 2.67E+03 0.0 0.1 0.00.1 dose2/Seed + Dry dai = days after inoculation (pre-inoculation). “/”= wet sequential method (the first products are applied and then theremaining products are applied onto the wet seed); “//” = dry sequentialmethod (the first products are applied and after several minutes theremaining products are applied onto the dry seed); “+” = simultaneousmethod (the products are mixed and after a few minutes the mixture isapplied onto the seed). UHC + Premax LLI dose1 = 3 mL highlyconcentrated Inoculant/kg + 1 mL Premax LLI/kg; UHC + Premax LLI dose2 =4 mL highly concentrated Inoculant/kg + 1 mL Premax LLI/kg.

Example 2

Survival of Bradyrhizobium japonicum on Soybean Seed and NodulationResponse in Treatments with Agrochemicals Used in the United States andCanada

This example used the highly concentrated inoculant after 2 yearsstorage since manufacture. In addition, its performance was evaluated incomparison with different commercial agrochemicals commonly used in theUnited States or Canada. The highly concentrated inoculant was appliedin 5 different doses and using different application methods. Theresults were evaluated according to the viability of the bacteria on thesoybean, the number of bacteria per seed; the nodulation response in theplant growth chamber, and the time since treatment (pre-inoculation).

The highly concentrated liquid inoculant used in this example wasmanufactured from Stabilizer A1 and contained 2.15×10¹¹ cfu/mL, with 2years since its manufacture. In some seed treatments, the right dose ofinoculant (UHC) was directly applied to the seed and in other cases, theinoculant was previously mixed with a bacterial protector (Premax); themix was let sit for 15 minutes and the proper dose was applied onto theseed. Each seed treatment involved 400 g of DM 3312 variety soybean,recently treated with commercial agrochemicals in widespread use in theUnited States and/or Canadian markets. Non-agrochemical controls werealso considered. Table 5 shows the products used and their applicationrates on the seeds.

TABLE 5 Dosage (quantity/kg Product name Product type Active ingredient(%) soybean seed) UHC Highly Bradyrhizobium japonicum + 0.98 mL, 0.73mL, concentrated Bradyrhizobium diazoefficiens 0.65 mL, 0.5 mL bacterial(15) or 0.48 mL inoculant Premax Bacterial Sugar (% not applicable) 0.5mL, 0.25 mL protector or 0.15 mL Intego Suite Fungicide Clothianidin(20.06) + ethaboxam  2.2 mL Soybeans (2.97) + ipconazole (0.99) +metalaxyl (0.79) CruiserMaxx Insecticide Thiamethoxam (20.8) + 2.11 mLVibrance and fungicide mefenoxam (3.13) + fludioxonil (1.04) + sedaxane(1.04)

Levels 34, 77, 120, 149, 181 and 210 days after inoculation (dai) wereestablished, corresponding to the time between the seed treatment andthe biological determinations (early seed treatment or pre-inoculation).Storage of the treated seed was in the temperature conditions closest tothose in the U.S. and Canadian warehouses, starting with the equivalentof seed treatment in November (fall) (Table 6).

TABLE 6 Storage temperature Storage time of treated of treated seed (°C.) seed (dai) 5  0 to 30 2  31 to 120 5 121 to 150 10 151 to 180 16 181to 210

The test results were measured in the same way as in the previousexample, extensively detailed in the section “Technics and Measurementof Results”. The test results can be seen graphically in FIG. 5 (cfu ofBradyrhizobium/seed), FIG. 6 (nodules per plant in primary root), FIG. 7(nodules per plant in total root), FIG. 8 (dry mass of nodules per plantin primary root) and FIG. 9 (dry mass of nodules per plant in totalroot). The highly concentrated bacterial inoculum (UHC) can be verifiedas effective since it responded in numerous cases with nodules per plantin total root and mg of dry mass of nodules in total root. Thesuitability was influenced by the degree of compatibility withagrochemicals, the pre-inoculation time and the inoculant dose. Asuitable response was possible even under the most aggressive conditionsfor bacteria, given by the 210 dai, the presence of agrochemicals andthe lower doses of inoculant (0.48 to 0.65 mL/kg). The best responseunder these conditions was observed in the following seed treatments:“Intego Suite Soybeans/UHC (0.48 mL/kg)+Premax (0.5 mL/kg)”, “IntegoSuite Soybeans/UHC (0.65 mL/kg)” and “CruiserMaxx Vibrance/UHC (0.65mL/kg)”, which produced ≥3.9 nodules per plant in total root and ≥15.1mg of dry mass of nodules in total root.

1. A highly concentrated bacterial liquid inoculant containing aconcentration of bacteria per volume unit between 10¹⁰ and 10¹¹ cfu/mL,with a shelf life of 2 years at storage temperatures up to 25° C., wherethe minimum concentration at expiry is 2×10¹⁰ cfu/mL.
 2. The highlyconcentrated bacterial liquid inoculant in accordance with claim 1 whosebacteria are selected from the Bradyrhizobium japonicum, B.dizoefficiens or B. elkanii group, preferably Bradyrhizobium japonicum.3. The highly concentrated bacterial liquid inoculant in accordance withclaim 1, where the storage temperature may range between 2 to 25° C. 4.The highly concentrated bacterial liquid inoculant in accordance withclaim 1, whose final formulation incorporates a stabilizer selectedfrom: A1, A1810 and A2.
 5. The highly concentrated bacterial liquidinoculant in accordance with claim 4, whose final formulationincorporates an A1 stabilizer.
 6. The highly concentrated bacterialliquid inoculant in accordance with claim 1, where the inoculant may beapplied onto the seed or in the sowing furrow.
 7. The highlyconcentrated bacterial liquid inoculant in accordance with claim 1,where the application on the seed must be applied with at least oneagrochemical product selected from: bacterial protector, nematicide,insecticide, fungicide, drying powder, nutritional supplement.
 8. Thehighly concentrated bacterial liquid inoculant in accordance with claim2, where the storage temperature may range between 2 to 25° C.
 9. Thehighly concentrated bacterial liquid inoculant in accordance with claim2, whose final formulation incorporates a stabilizer selected from: A1,A1810 and A2.
 10. The highly concentrated bacterial liquid inoculant inaccordance with claim 9, whose final formulation incorporates an A1stabilizer.
 11. The highly concentrated bacterial liquid inoculant inaccordance with claim 2, where the inoculant may be applied onto theseed or in the sowing furrow.
 12. The highly concentrated bacterialliquid inoculant in accordance with claim 3, where the inoculant may beapplied onto the seed or in the sowing furrow.
 13. The highlyconcentrated bacterial liquid inoculant in accordance with claim 4,where the inoculant may be applied onto the seed or in the sowingfurrow.
 14. The highly concentrated bacterial liquid inoculant inaccordance with claim 2, where the application on the seed must beapplied with at least one agrochemical product selected from: bacterialprotector, nematicide, insecticide, fungicide, drying powder,nutritional supplement.
 15. The highly concentrated bacterial liquidinoculant in accordance with claim 3, where the application on the seedmust be applied with at least one agrochemical product selected from:bacterial protector, nematicide, insecticide, fungicide, drying powder,nutritional supplement.
 16. The highly concentrated bacterial liquidinoculant in accordance with claim 4, where the application on the seedmust be applied with at least one agrochemical product selected from:bacterial protector, nematicide, insecticide, fungicide, drying powder,nutritional supplement.
 17. The highly concentrated bacterial liquidinoculant in accordance with claim 5, where the application on the seedmust be applied with at least one agrochemical product selected from:bacterial protector, nematicide, insecticide, fungicide, drying powder,nutritional supplement.